Symbiosis
Symbiosis has played a key role in the evolution of life on Earth. Symbiotic mergers of once independent species drove the origin of eukaryotes. Moreover, symbiosis has enabled many species to gain novel functions and occupy new ecological niches, thus underpinning the functioning of diverse ecosystems. As endosymbionts, microbes provide their eukaryotic hosts with an array of ecological and physiological innovations, including new metabolic capabilities, such as autotrophy or nitrogen fixation, and protection against infections or environmental stressors. Microbial eukaryotes also commonly host their own endosymbionts, including bacteria and algae. Understanding the stability and resilience of symbioses is key to predicting the response of important ecosystems, such as coral reefs, to global change. Manipulating symbiotic associations also has far-reaching economic, environmental and medical implications, through the potential to improve crop productivity, reduce reliance on fertilisers, and control the insect vectors of infectious diseases.
This collection, guest edited by Professor Michael Brockhurst (University of Manchester) and Dr. Rebecca J Hall (University of Birmingham), will feature microbe-focused studies of symbiosis, ranging from the molecular mechanisms of host-symbiont interactions, their genetic and genomic diversity, to understanding the impacts of symbioses in natural and manmade ecosystems.
Collection Contents
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Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti
In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.
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Genetic and biochemical characterization of arginine biosynthesis in Sinorhizobium meliloti 1021
l-Ornithine production in the alfalfa microsymbiont Sinorhizobium meliloti occurs as an intermediate step in arginine biosynthesis. Ornithine is required for effective symbiosis but its synthesis in S. meliloti has been little studied. Unlike most bacteria, S. meliloti 1021 is annotated as encoding two enzymes producing ornithine: N-acetylornithine (NAO) deacetylase (ArgE) hydrolyses NAO to acetate and ornithine, and glutamate N-acetyltransferase (ArgJ) transacetylates l-glutamate with the acetyl group from NAO, forming ornithine and N-acetylglutamate (NAG). NAG is the substrate for the second step of arginine biosynthesis catalysed by NAG kinase (ArgB). Inactivation of argB in strain 1021 resulted in arginine auxotrophy. The activity of purified ArgB was significantly inhibited by arginine but not by ornithine. The purified ArgJ was highly active in NAO deacetylation/glutamate transacetylation and was significantly inhibited by ornithine but not by arginine. The purified ArgE protein (with a 6His-Sumo affinity tag) was also active in deacetylating NAO. argE and argJ single mutants, and an argEJ double mutant, are arginine prototrophs. Extracts of the double mutant contained aminoacylase (Ama) activity that deacetylated NAO to form ornithine. The purified products of three candidate ama genes (smc00682 (hipO1), smc02256 (hipO2) and smb21279) all possessed NAO deacetylase activity. hipO1 and hipO2, but not smb21279, expressed in trans functionally complemented an Escherichia coli ΔargE : : Km mutant. We conclude that Ama activity accounts for the arginine prototrophy of the argEJ mutant. Transcriptional assays of argB, argE and argJ, fused to a promoterless gusA gene, showed that their expression was not significantly affected by exogenous arginine or ornithine.
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Glycerol utilization by Rhizobium leguminosarum requires an ABC transporter and affects competition for nodulation
More LessPlasmid curing has shown that the ability to use glycerol as a carbon source is plasmid-encoded in Rhizobium leguminosarum. We isolated the locus responsible for glycerol utilization from plasmid pRleVF39c in R. leguminosarum bv. viciae VF39. This region was analyzed by DNA sequencing and mutagenesis. The locus encompasses a gene encoding GlpR (a DeoR regulator), genes encoding an ABC transporter, and genes glpK and glpD, encoding a kinase and dehydrogenase, respectively. All the genes except the regulatory gene glpR were organized into a single operon, and were required for growth on glycerol. The glp operon was strongly induced by both glycerol and glycerol 3-phosphate, as well as by pea seed exudate. GlpR repressed the operon in the absence of inducer. Mutation of genes encoding the ABC transporter abolished all transport of glycerol in transport assays using radiolabelled glycerol. This confirms that, unlike in other organisms such as Escherichia coli and Pseudomonas aeruginosa, which use facilitated diffusion, glycerol uptake occurs by an active process in R. leguminosarum. Since the glp locus is highly conserved in all sequenced R. leguminosarum and Rhizobium etli strains, as well as in Sinorhizobium spp. and Agrobacterium spp. and other alphaproteobacteria, this process for glycerol uptake is probably widespread. Mutants unable to use glycerol were deficient in competitiveness for nodulation of peas compared with the wild-type, suggesting that glycerol catabolism confers an advantage upon the bacterium in the rhizosphere or in the infection thread.
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Gene expression level influences amino acid usage, but not codon usage, in the tsetse fly endosymbiont Wigglesworthia
More LessWigglesworthia glossinidia brevipalpis, the obligate bacterial endosymbiont of the tsetse fly Glossina brevipalpis, is characterized by extreme genome reduction and AT nucleotide composition bias. Here, multivariate statistical analyses are used to test the hypothesis that mutational bias and genetic drift shape synonymous codon usage and amino acid usage of Wigglesworthia. The results show that synonymous codon usage patterns vary little across the genome and do not distinguish genes of putative high and low expression levels, thus indicating a lack of translational selection. Extreme AT composition bias across the genome also drives relative amino acid usage, but predicted high-expression genes (ribosomal proteins and chaperonins) use GC-rich amino acids more frequently than do low-expression genes. The levels and configuration of amino acid differences between Wigglesworthia and Escherichia coli were compared to test the hypothesis that the relatively GC-rich amino acid profiles of high-expression genes reflect greater amino acid conservation at these loci. This hypothesis is supported by reduced levels of protein divergence at predicted high-expression Wigglesworthia genes and similar configurations of amino acid changes across expression categories. Combined, the results suggest that codon and amino acid usage in the Wigglesworthia genome reflect a strong AT mutational bias and elevated levels of genetic drift, consistent with expected effects of an endosymbiotic lifestyle and repeated population bottlenecks. However, these impacts of mutation and drift are apparently attenuated by selection on amino acid composition at high-expression genes.
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Glutamate Synthase Activity in Symbiotic Cyanobacteria
More LessGlutamate synthase (GOGAT) activities of the symbiotic cyanobacteria of the lichens Peltigera canina and Peltigera aphthosa and of the water fern Azolla caroliniana have been determined and, like glutamine synthetase (GS) activity, found to be substantially reduced compared with the activities found in the free-living cyanobacteria. A similar reduction in GOGAT activity was not noted in the symbiotic green alga (Coccomyxa sp.) in P. aphthosa. The selective reduction of GS-GOGAT activity in symbiosis may be related to the production of extracellular nitrogen by the symbiotic cyanobacterium.
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Genetic Relationships among Bacterial Endosymbionts of Paramecium aurelia
More LessGenetic relationships among bacterial endosymbionts of Paramecium assigned to the genus Caedobacter Preer et al. 1974 were determined using DNA-DNA hybridization techniques. It was shown that mu, nu and pi endosymbionts are closely related to each other, but not to any of the strains of kappa endosymbionts tested, and that kappa consists of at least three distinct and genetically diverse groups. Therefore, it is proposed that Caedobacter be split into two genera, Caedobacter and Pseudocaedobacter, depending upon the ability or lack of ability, respectively, to produce cells containing R bodies. Strains were screened for covalently closed, circular DNA by ethidium bromide/CsCl centrifugation, but only pi and hump-killer kappa particles were found to contain this form of DNA.
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